Prostate cancer is a leading cause of morbidity and mortality for men in the industrialised world. Degarelix, also known as FE200486, is a third generation gonadotropin releasing hormone (GnRH) receptor antagonist (a GnRH blacker) that has been developed and recently approved for prostate cancer patients in need of androgen ablation therapy (Doehn et al., Drugs 2006, vol. 9, No. 8, pp. 565-571; WO 09846634). Degarelix acts by immediate and competitive blockade of GnRH receptors in the pituitary and, like other GnRH antagonists, does not cause an initial stimulation of luteinizing hormone production via the hypothalamic-pituitary-gonadal axis, and therefore does not cause testosterone surge or clinical flare (Van Poppel, Cancer Management and Research, 2010:2 39-52; Van Poppel et al., Urology, 2008, 71(6), 1001-1006); James, E. F. et al., Drugs, 2009, 69(14), 1967-1976).
Degarelix is a synthetic linear decapeptide containing seven unnatural amino acids, five of which are D-amino acids. It has ten chiral centers in the back bone of the decapeptide. The amino acid residue at position 5 in the sequence has an additional chiral center in the side-chain substitution giving eleven chiral centers in total. Its CAS registry number is 214766-78-6 (of free base) and it is commercially available under the Trademark Firmagon™. The drug substance is chemically designated as D-Alaninamide, N-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L-seryl-4-[[[(4S)-hexahydro-2,6-diaxo-4-pyrimidinyl]carbonyl]amino]-L-phenylalanyl-4-[(aminocarbonyl)amino]-D-phenylalanyl-L-leucyl-N6-(1-methylethyl)-L-lysyl-L-prolyl- and is represented by the chemical structure below:

The structure of Degarelix can also be represented as:Ac-D-2NaI-D-4Cpa-D-3PaI-Ser-4Aph(L-Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 where Ac is acetyl, 2NaI is 2-naphthylalanine, 4 Cpa is 4-chlorophenylalanine, 3 PaI is 3-pyridylalanine, Ser is serine, 4Aph is 4-aminophenylalanine, Hor is hydroorotyl, Cbm is carbamoyl, Leu is leucine, Lys(iPr) is N6-isopropyllysine, Pro is proline and Ala is alanine.
For the purposes of describing this invention, each amino acid in Degarelix will be given the shorthand notation as follows:
AA1 is D-2NaI, AA2 is D-4 Cpa, AA3 is D-3 PaI, AA4 is Ser, AA5 is 4Aph(L-Hor), AA6 is D-Aph(Cbm), AA7 is Leu, AA8 is Lys(iPr), AA9 is Pro and AA10 is D-Ala.
Thus, as an example, Degarelix can be represented as Ac-AA1-AA10-NH2, the tetrapeptide Ac-D-2NaI-D-4 Cpa-D-3 PaI-Ser can be represented as Ac-AA1-AA4 and the hexapeptide 4Aph(L-Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 as AA5-AA10-NH2.
Degarelix has previously been prepared using Boc-solid phase peptide synthesis (SPPS) methodology as reported in WO 98/46634 and Jiang et al., J. Med. Chem. 2001, 44, 453-467. Basically, Boc-protected D-Ala is first coupled to MBNA resin in dimethylformamide (DMF)/CH2Cl2 using diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) as activating or coupling agents. Once D-Ala is coupled to the resin, synthesis proceeds by washing, deblocking and then coupling the next amino acid residue until the decapeptide has been completed. The side chain primary amino groups of 4Aph in the 5-position and of D-4Aph in the 6-position are protected by Fmoc when they are added and modified with L-Hor and Cbm respectively before the next amino acid in the chain is added. This requires the additional steps of first removing the side-chain protection with piperidine, reacting the newly freed amino group on the peptidoresin with tert-butyl isocyanate or L-hydroorotic acid, ensuring that the reaction is complete with a ninhydrin test and then washing the peptidoresin before adding the next amino acid residue (see also Sorbera et al., Drugs of the Future 2006, Vol. 31, No. 9, pp 755-766).
While Boc-SPPS methodology has afforded sufficient quantities of Degarelix until now, the growing demand for this polypeptide means that ever larger quantities are required. Boc-SPPS, which requires HF cleavage, is not suited to large scale industrial synthesis. Indeed, WO 98/46634 mentions that SPPS is only suitable for limited quantities of up to 1 kg while classical peptide solution synthesis, or liquid phase peptide synthesis (LPPS), is preferred for larger quantities of product. WO 98/46634 does not specify how such synthesis should be performed. Further, expense attributable the large excess of coupling reagents, additives, and amino acids required for the SPPS. While the existence of a liquid phase peptide synthesis of Degarelix has been reported [EMEA Report: Assessment Report for Firmagon™ (Degarelix): Doc. Ref. EMEA/CHMP/635761/2008], as of now no details of such a process have been publically disclosed.
WO 97/34923 and WO 99/26964 are international Application Publications which are concerned with liquid phase processes for the preparation of biologically active peptides. WO 99/26964 is particularly concerned with the liquid phase synthesis of decapeptides having activity as GnRH antagonists. WO 99/26964 lists a number of inherent limitations of the SPPS methodology for producing GnRH antagonists including the limited capacity of the resin, the large excess of reagents and amino acids required, as well as the need to protect all reactive side chains such as the hydroxy group in Ser, the aromatic amino groups in Aph and D-Aph, the ε-i-propylamino group in Lys(i-Pr).
International Application Publication No. WO 99/26964 describes a liquid phase process which involves first preparing the central peptide fragments of the 5 and 6 positions of a decapeptide with the side chains fully elaborated and then assembling the peptide through a “4-2-4”, “3-3-4” or “3-4-3” fragment assembly pattern. For example, in the preparation of the GnRH antagonist Azaline B, a tetrapeptide is coupled with a hexapeptide to form the desired decapeptide. When the same fragment assembly pattern is attempted for Degarelix, racemisation of the Serine amino acid (AA4) occurs resulting in about 20% impurity of L-Ser. This impurity carries over into the final decapeptide and is difficult to remove. Furthermore, when preparing the tetrapeptide AA1-AA4 by adding the Ser unit to the tripeptide AA1-AA3 following the procedure described in WO 99/26964, tetrabutylammonium ions from the hydrolysis of the benzyl ester group could not be removed completely during the subsequent operations and were carried through to the final product. It was further found that in the Degarelix synthesis, the L-hydroorotyl group rearranges to its hydantainacetyl analogue when L-dihydroorotic acid is coupled with 4 Amp to prepare AA5. These and other problems with the solution-phase synthesis of Degarelix have now been overcome and a new solution-phase polypeptide synthesis of this decapeptide is disclosed herein for the first time.